US9913891B2 - FMDV and E2 fusion proteins and uses thereof - Google Patents

FMDV and E2 fusion proteins and uses thereof Download PDF

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US9913891B2
US9913891B2 US15/353,727 US201615353727A US9913891B2 US 9913891 B2 US9913891 B2 US 9913891B2 US 201615353727 A US201615353727 A US 201615353727A US 9913891 B2 US9913891 B2 US 9913891B2
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fmdv
polypeptide
protein
antigen
sequence
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US20170143819A1 (en
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Jean-Christophe Audonnet
Frédéric Reynard
Natalia Bomchil
Cécile Sigoillot-Claude
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Boehringer Ingelheim Animal Health USA Inc
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Merial Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32171—Demonstrated in vivo effect

Definitions

  • the present invention relates to compositions for combating Foot and Mouth Disease Virus (FMDV) infection in animals.
  • the present invention provides pharmaceutical compositions comprising an FMDV antigen, methods of vaccination against the FMDV, methods of producing antigens, and kits for use with such methods and compositions.
  • Foot-and-mouth disease is one of the most virulent and contagious diseases affecting farm animals. This disease is endemic in numerous countries in the world, especially in Africa, Asia and South America. In addition, epidemic outbreaks can occur periodically. The presence of this disease in a country may have very severe economic consequences resulting from loss of productivity, loss of weight and milk production in infected herds, and from trade embargoes imposed on these countries.
  • the measures taken against this disease consist of strict application of import restrictions, hygiene controls and quarantine, slaughtering sick animals and vaccination programs using inactivated vaccines, either as a preventive measure at the national or regional level, or periodically when an epidemic outbreak occurs.
  • FMD is characterized by its short incubation period, its highly contagious nature, the formation of ulcers in the mouth and on the feet and sometimes, the death of young animals. FMD affects a number of animal species, in particular cattle, pigs, sheep and goats.
  • the agent responsible for this disease is a ribonucleic acid (RNA) virus belonging to the Aphthovirus genus of the Picornaviridae family (Cooper et al., 1978, Intervirology 10, 165-180).
  • RNA ribonucleic acid
  • At present, at least seven types of foot-and-mouth disease virus (FMDV) are known: the European types (A, O and C), the African types (SAT1, SAT2 and SAT3) and an Asiatic type (Asia 1). Numerous sub-types have also been distinguished (Kleid et al., 1981, Science 214, 1125-1129).
  • FMDV is a naked icosahedral virus of about 25 nm in diameter, containing a single-stranded RNA molecule consisting of about 8500 nucleotides, with a positive polarity.
  • This RNA molecule comprises a single open reading frame (ORF), encoding a single polyprotein containing, inter alia, the capsid precursor also known as protein P1 or P88.
  • the protein P1 is myristylated at its amino-terminal end. During the maturation process, the protein P1 is cleaved by the protease 3C into three proteins known as VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively; Belsham G.
  • the protein VP0 is then cleaved into two proteins, VP4 and VP2 (or 1A and 1B respectively).
  • the mechanism for the conversion of the proteins VP0 into VP1 and VP3, and for the formation of mature virions is not known.
  • the proteins VP1, VP2 and VP3 have a molecular weight of about 26,000 Da, while the protein VP4 is smaller at about 8,000 Da.
  • Pyruvate dehydrogenase (PDH) complexes are multifunctional enzymes that contain three essential enzymes: a thiamine-dependent pyruvate decarboxylase (E1), a dihydrolipoyl acetyltransferase (E2), and a flavoprotein dihydrolipoyl dehydrogenase (E3) (Lengyel et al., Structure, 16:93-103, 2008).
  • E1 thiamine-dependent pyruvate decarboxylase
  • E2 dihydrolipoyl acetyltransferase
  • E3 flavoprotein dihydrolipoyl dehydrogenase
  • compositions comprising an antigenic FMDV polypeptide and fragments and variants thereof are provided.
  • the FMDV antigens and fragments and variants thereof possess immunogenic and protective properties.
  • the FMDV antigens may be produced as a fusion protein with E2 from Geobacillus stearothermophilus .
  • the FMDV fusion proteins of the present invention assemble into particles which resemble the natural FMDV virions or VLPs.
  • the antigenic polypeptides and fragments and variants thereof can be formulated into vaccines and/or pharmaceutical compositions.
  • Such vaccines can be used to vaccinate an animal and provide protection against at least one FMDV strain.
  • Methods of the invention include methods for making the antigenic polypeptides.
  • Methods include methods for making antigens or antigenic polypeptides fused to E2 from Geobacillus stearothermophilus .
  • the invention provides a method to construct conformationally correct immunogens lacking the infective FMDV genome to make effective and safe vaccines.
  • the methods facilitate the simple and easy expression of FMDV antigens in vitro which replicate and simulate the conformation of the natural and authentic FMDV virions or VLPs that are complex and difficult to produce.
  • Methods also include methods of use including administering to an animal an effective amount of an antigenic polypeptide or fragment or variant thereof to produce a protective immunogenic response. After production, the antigenic polypeptide can be partially or substantially purified for use as a vaccine.
  • Kits comprising at least one antigenic polypeptide or fragment or variant thereof and instructions for use are also provided.
  • FIG. 1 depicts a table summarizing the DNA and Protein sequences.
  • FIGS. 2A-2F depict the DNA and proteins sequences, and sequence alignments.
  • FIG. 3 depicts the Western blot and dot blot of the expressed FMDV-E2 fusion protein.
  • FIGS. 4A-4C depict the production of FMDV-E2 particles.
  • FIG. 5 depicts the indirect ELISA of E2-FMDV particles.
  • FIG. 6 depicts the plasma cells results on D27 of O1M vaccines.
  • FIG. 7 depicts the plasma cells results on D42 of O1M vaccines.
  • FIG. 8 depicts the IFN ⁇ ELISPOT on D27 of O1M vaccine.
  • FIG. 9 depicts the IFN ⁇ ELISPOT on D42 of O1M vaccine.
  • FIG. 10 depicts the memory B cells on D42 of O1M vaccine.
  • FIG. 11 depicts the FMDV O1 Manisa SN.
  • compositions comprising an FMDV polypeptide, antigen and fragments and variants thereof that elicit an immunogenic response in an animal are provided.
  • the antigenic polypeptides or fragments or variants thereof are produced as fusion proteins or chimeric proteins with E2 from Geobacillus stearothermophilus .
  • the antigenic polypeptides or fragments or variants may be formulated into vaccines or pharmaceutical compositions and used to elicit or stimulate a protective response in an animal.
  • the polypeptide antigen is an FMDV synthetic antigens VP1 or active fragment or variant thereof.
  • the antigenic polypeptides or antigens of the invention may be full length polypeptides or active fragments or variants thereof.
  • active fragments or “active variants” is intended that the fragments or variants retain the antigenic nature of the polypeptide.
  • the present invention encompasses any FMDV polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal.
  • the FMDV polypeptide, antigen, epitope or immunogen may be any FMDV polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in an animal, such as an ovine, bovine, caprine or swine.
  • FMDV antigenic polypeptides include VP1 of FMDV.
  • FMDV is a naked icosahedral virus of about 25 nm in diameter, containing a single-stranded RNA molecule consisting of about 8500 nucleotides, with a positive polarity.
  • This RNA molecule comprises a single open reading frame (ORF), encoding a single polyprotein containing, inter alia, the capsid precursor also known as protein P1 or P88.
  • the protein P1 is myristylated at its amino-terminal end.
  • the protein P1 is cleaved by the protease 3C into three proteins known as VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J., Progress in Biophysics and Molecular Biology, 1993, 60, 241-261).
  • the protein VP0 is then cleaved into two proteins, VP4 and VP2 (or 1A and 1B respectively).
  • the mechanism for the conversion of the proteins VP0 into VP1 and VP3, and for the formation of mature virions is not known.
  • the proteins VP1, VP2 and VP3 have a molecular weight of about 26,000 Da, while the protein VP4 is smaller at about 8,000 Da.
  • the simple combination of the capsid proteins forms the protomer or 5S molecule, which is the elementary constituent of the FMDV capsid. This protomer is then compiled into a pentamer to form the 12S molecule.
  • the virion results from the encapsidation of a genomic RNA molecule by assembly of twelve 12S pentamers, thus constituting the 146S particles.
  • the viral capsid may also be formed without the presence of an RNA molecule inside it (hereinafter “empty capsid”).
  • the empty capsid is also designated as particle 75S.
  • the formation of empty capsids may occur naturally during viral replication or may be produced artificially by chemical treatment.
  • the present invention relates to bovine, ovine, caprine, or swine vaccines or compositions which may comprise an effective amount of an FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle.
  • the vaccines further comprise adjuvants, such as the oil-in-water (O/W) emulsions described in U.S. Pat. No. 7,371,395.
  • adjuvants such as the oil-in-water (O/W) emulsions described in U.S. Pat. No. 7,371,395.
  • the adjuvants include EMULSIGEN, Aluminum Hydroxide and Saponin, and CpG, or combinations thereof.
  • the response in the animal is a protective immune response.
  • animal it is intended mammals, birds, and the like.
  • Animal or host includes mammals, artiodactyles and human.
  • the animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), swine (e.g., pig), caprine (e.g., goat), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and fish.
  • the antigenic polypeptides of the invention are capable of protecting against FMDV. That is, they are capable of stimulating an immune response in an animal.
  • antigen or “immunogen” means a substance that induces a specific immune response in a host animal.
  • the antigen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof.
  • the immunogen or antigen may comprise a toxin or antitoxin.
  • immunogenic protein, polypeptide, or peptide includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein.
  • the protein fragment is such that it has substantially the same immunological activity as the total protein.
  • a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant.
  • An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof.
  • immunological response is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above.
  • fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996).
  • epitope mapping techniques well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996).
  • linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • the term “immunogenic protein, polypeptide, or peptide” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
  • conservative variation denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue.
  • particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity.
  • Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein.
  • the term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
  • epitope refers to the site on an antigen or hapten to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”.
  • Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • an “immunological response” to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest.
  • an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
  • Immunogenic fragments for purposes of the present invention, will usually include at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.
  • a minimum structure of a polynucleotide expressing an epitope is that it comprises or consists essentially of or consists of nucleotides encoding an epitope or antigenic determinant of an FMDV polypeptide.
  • a polynucleotide encoding a fragment of an FMDV polypeptide may comprise or consist essentially of or consist of a minimum of 15 nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or 150 consecutive or contiguous nucleotides of the sequence encoding the polypeptide.
  • nucleic acid and “polynucleotide” refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
  • the sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component.
  • Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support.
  • the polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
  • genes are used broadly to refer to any segment of polynucleotide associated with a biological function.
  • genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression.
  • gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
  • protein protein
  • peptide polypeptide
  • polypeptide fragment polymers of amino acid residues of any length.
  • the polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • fusion protein or “chimeric protein” as used herein refers to any recombinant protein created through the joining of two or more heterologous genes. The expression of the heterologous genes results in one single polypeptide with functional properties delivered from each of the original proteins.
  • an “isolated” biological component refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
  • a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide in its natural environment. That is the polypeptide is separated from cellular components.
  • substantially purified it is intended that such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed.
  • the polypeptide may be partially purified.
  • partially purified is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides.
  • the polypeptides disclosed herein can be purified by any of the means known in the art.
  • the antigenic polypeptides or fragments or variants thereof are FMDV antigenic polypeptides that are produced as fusion proteins. Fragments and variants of the disclosed polynucleotides and polypeptides encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the antigenic amino acid sequence encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have immunogenic activity as noted elsewhere herein. Fragments of the polypeptide sequence retain the ability to induce a protective immune response in an animal.
  • a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • Variants of a particular polynucleotide of the invention can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • “Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is they the ability to elicit an immune response.
  • the present invention provides FMDV polypeptides from ovine, bovine, caprine, or swine. In another aspect, the present invention provides a polypeptide having a sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12 and 14, and variant or fragment thereof.
  • homologs of FMDV polypeptides from ovine, bovine, caprine, or swine are intended to be within the scope of the present invention.
  • the term “homologs” includes orthologs, analogs and paralogs.
  • the term “analogs” refers to two polynucleotides or polypeptides that have the same or similar function, but that have evolved separately in unrelated organisms.
  • the term “orthologs” refers to two polynucleotides or polypeptides from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the same or similar functions.
  • allelic variants refers to two polynucleotides or polypeptides that are related by duplication within a genome. Paralogs usually have different functions, but these functions may be related. Analogs, orthologs, and paralogs of a wild-type FMDV polypeptide can differ from the wild-type FMDV polypeptide by post-translational modifications, by amino acid sequence differences, or by both. In particular, homologs of the invention will generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence identity, with all or part of the wild-type FMDV or polynucleotide sequences, and will exhibit a similar function. Variants include allelic variants.
  • allelic variant refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1-5% variance in a polynucleotide or a polypeptide. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same gene genetic locus in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention.
  • the FMDV antigen is fused to an E2 of Geobacillus stearothermophilus.
  • the term “derivative” or “variant” refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide.
  • variants or derivatives include polypeptides having minor modifications of the FMDV polypeptide primary amino acid sequences that may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.
  • variant further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
  • conservative variation denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue.
  • particularly preferred substitutions will generally be conservative in nature, as described above.
  • polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, e.g., optimized codon usage for a specific host.
  • optimized refers to a polynucleotide that is genetically engineered to increase its expression in a given species.
  • the DNA sequence of the FMDV protein gene can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucleotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites.
  • Increased expression of FMDV protein in said species can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species.
  • frequency of preferred codon usage refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the FMDV polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • sequence identity between two amino acid sequences may be established by the NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using the standard parameters (see, e.g., the BLAST or BLASTX algorithm available on the “National Center for Biotechnology Information” (NCBI, Bethesda, Md., USA) server, as well as in Altschul et al.; and thus, this document speaks of using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term “blasts”).
  • the “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., IntelligeneticsTM Suite, Intelligenetics Inc. CA).
  • IntelligeneticsTM Suite Intelligenetics Inc. CA
  • RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
  • sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucleotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, Calif.).
  • Hybridization reactions can be performed under conditions of different “stringency.” Conditions that increase stringency of a hybridization reaction are well known. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 2014).
  • the invention further encompasses the FMDV polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer.
  • a “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo.
  • the heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette.
  • a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.
  • recombinant means a polynucleotide semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
  • Heterologous means derived from a genetically distinct entity from the rest of the entity to which it is being compared.
  • a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
  • the present invention relates to ovine, bovine, caprine and swine vaccines or pharmaceutical or immunological compositions which may comprise an effective amount of FMDV antigens and a pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle.
  • the present invention relates to a FMDV vaccine or composition which may comprise an effective amount of an FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle.
  • the FMDV antigen is expressed as a fusion protein or chimeric protein with E2 from Geobacillus stearothermophilus.
  • the subject matter disclosed herein is directed to a fusion or chimeric protein comprising an FMDV antigen.
  • the subject matter disclosed herein is directed to a vaccine or composition comprising an FMDV antigen fused to E2 from Geobacillus stearothermophilus.
  • the subject matter disclosed herein is directed to a vaccine or composition comprising an FMDV and E2 fusion or chimeric protein produced in prokaryotes or eukaryotes.
  • the present invention encompasses any FMDV polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal, such as an ovine, bovine, caprine or porcine.
  • the FMDV polypeptide, antigen, epitope or immunogen may be any FMDV polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment thereof, that elicits, induces or stimulates a response in an animal, such as an ovine, bovine, caprine or porcine.
  • the FMDV antigen may be a synthetic antigen designed from sequence alignment. Multiple B and T epitopes were described in FMD VP1 capsid protein. A synthetic polypeptide corresponding to the fusion of one T epitope and two B epitopes from Asia serotype have been proved to induce a protective response (Ren et al., Vaccine 2011). After sequence alignment, an equivalent polypeptide could be defined for all other serotypes.
  • the FMDV antigen may be a VP1 polypeptide.
  • the FMDV antigen may be a P1-3C polypeptide.
  • the FMDV antigen may be P1 alone, or P1-2A/2B1.
  • the FMDV antigen may be VP0-VP3.
  • the FMDV antigen may be VP4-VP2.
  • the FMDV antigen may be 3C.
  • the FMDV antigen or polypeptide or protein is a fusion or chimeric protein. In yet another embodiment, the FMDV antigen or polypeptide or protein is fused to the core C-terminal catalytic domain of E2 from Geobacillus stearothermophilus.
  • the FMDV antigen may be derived from FMDV strains O1 Manisa, O1 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran'96, A22 Iraq, SAT2 Saudi Arabia.
  • the present invention relates to an FMDV vaccine which may comprise an effective amount of a recombinant or fusion or chimeric FMDV antigen or protein and a pharmaceutically or veterinarily acceptable carrier, adjuvant, excipient, or vehicle.
  • the FMDV antigen is a fusion or chimeric FMDV and E2 protein.
  • the FMDV-E2 fusion or chimeric protein may be produced in prokaryotes or eukaryotes.
  • pharmaceutically or veterinarily acceptable carrier, adjuvant, excipient, or vehicle may be a water-in-oil emulsion.
  • pharmaceutically or veterinarily acceptable carrier, adjuvant, excipient, or vehicle may be an oil-in-water emulsion.
  • the water-in-oil emulsion may be a water/oil/water (W/O/W) triple emulsion.
  • the invention further encompasses the FMDV polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer.
  • the present invention provides FMDV polypeptides, particularly ovine, bovine, caprine or swine polypeptides having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, and variant or fragment thereof.
  • the present invention provides a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to an antigenic polypeptide of the invention, particularly to the polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14.
  • the present invention provides fragments and variants of the FMDV polypeptides identified above (SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14) which may readily be prepared by one of skill in the art using well-known molecular biology techniques.
  • the present invention provides a fusion or chimeric protein comprising FMDV antigen and E2 domain from Geobacillus stearothermophilus .
  • the FMDV-E2 fusion or chimeric protein may comprise a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having the sequence as set forth in SEQ ID NO:6, 10 or 14.
  • Variants are homologous polypeptides having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14.
  • An immunogenic fragment of an FMDV polypeptide includes at least 8, 10, 15, or 20 consecutive amino acids, at least 21 amino acids, at least 23 amino acids, at least 25 amino acids, or at least 30 amino acids of an FMDV polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14, or variants thereof.
  • a fragment of an FMDV polypeptide includes a specific antigenic epitope found on an FMDV polypeptide.
  • the present invention provides a polynucleotide encoding an FMDV polypeptide, such as a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14.
  • the present invention provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14, or a conservative variant, an allelic variant, a homolog or an immunogenic fragment comprising at least eight or at least ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.
  • the present invention provides a polynucleotide encoding an FMDV-E2 fusion or chimeric protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14.
  • the present invention provides a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, and 13, or a variant thereof.
  • the present invention provides a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having a sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, and 13, or a variant thereof.
  • the present invention provides E2 protein (SEQ ID NO:4) and DNA (SEQ ID NO:3) sequences, and E2 linker sequence (SEQ ID NO:15).
  • the polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • additional sequences such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • an FMDV polypeptide, antigen, epitope or immunogen are advantageously present in an inventive vector.
  • this comprises, consists essentially of, or consists of an initiation codon (ATG), a stop codon and a promoter, and optionally also a polyadenylation sequence for certain vectors such as plasmid and certain viral vectors, e.g., viral vectors other than poxviruses.
  • ATG initiation codon
  • viral vectors e.g., viral vectors other than poxviruses.
  • the polynucleotide encodes a polyprotein fragment, e.g. an FMDV peptide
  • an ATG is placed at 5′ of the reading frame and a stop codon is placed at 3′.
  • Other elements for controlling expression may be present, such as enhancer sequences, stabilizing sequences, such as intron and signal sequences permitting the secretion of the protein.
  • the present invention also relates to compositions comprising vectors, such as expression vectors, e.g., therapeutic compositions.
  • the compositions can comprise one or more vectors, e.g., expression vectors, such as in vivo expression vectors, comprising and expressing one or more FMDV polypeptides, antigens, epitopes or immunogens.
  • the vector contains and expresses a polynucleotide that comprises, consists essentially of, or consists of a polynucleotide coding for (and advantageously expressing) an FMDV antigen, epitope or immunogen, in a pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle.
  • the other vector or vectors in the compositions comprise, consist essentially of or consist of a polynucleotide that encodes, and under appropriate circumstances the vector expresses one or more other proteins of an FMDV polypeptide, antigen, epitope or immunogen, or a fragment thereof.
  • the vector or vectors comprise, or consist essentially of, or consist of polynucleotide(s) encoding E2 from Geobacillus stearothermophilus.
  • the expression vector is a plasmid vector or a DNA plasmid vector, in particular an in vivo expression vector in prokaryotes or eukaryotes.
  • the pET30a, pET30b and pET30c can be utilized as a vector for the insertion of a polynucleotide sequence.
  • the pET-30a-c vectors carry a T7 promoter and an N-terminal His•Tag/thrombin/S•Tag/enterokinase configuration plus an optional C-terminal His•Tag sequence.
  • the pVR1020 or 1012 plasmid can be utilized as a vector for the insertion of a polynucleotide sequence.
  • the pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal sequence.
  • the human tPA signal comprises from amino acid M(1) to amino acid S(23) in Genbank under the accession number HUMTPA14.
  • the plasmid utilized as a vector for the insertion of a polynucleotide sequence can contain the signal peptide sequence of equine IGF1 from amino acid M(24) to amino acid A(48) in Genbank under the accession number U28070. Additional information on DNA plasmids which may be consulted or employed in the practice are found, for example, in U.S. Pat. Nos. 6,852,705; 6,818,628; 6,586,412; 6,576,243; 6,558,674; 6,464,984; 6,451,770; 6,376,473 and 6,221,362.
  • plasmid covers any DNA transcription unit comprising a polynucleotide according to the invention and the elements necessary for its in vivo expression in a cell or cells of the desired host or target; and, in this regard, it is noted that a supercoiled or non-supercoiled, circular plasmid, as well as a linear form, are intended to be within the scope of the invention.
  • Each plasmid comprises or contains or consists essentially of, in addition to the polynucleotide encoding an FMDV antigen, epitope or immunogen, optionally fused with a heterologous peptide sequence, variant, analog or fragment, operably linked to a promoter or under the control of a promoter or dependent upon a promoter.
  • a strong promoter functional in eukaryotic cells.
  • the strong promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig, the Super promoter (Ni, M. et al., Plant J.
  • the CMV-IE promoter can comprise the actual promoter part, which may or may not be associated with the enhancer part. Reference can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615, as well as to PCT Application No WO87/03905.
  • the CMV-IE promoter is advantageously a human CMV-IE (Boshart et al., 1985, Cell 41(2): 521-30) or murine CMV-IE.
  • the promoter has either a viral, a plant, or a cellular origin.
  • a strong viral promoter other than CMV-IE that may be usefully employed in the practice of the invention is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus.
  • a strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa et al., 2000, Vaccine 18(22): 2337-44), or the actin promoter (Miyazaki et al., 1989, Gene 79(2): 269-77).
  • polyA polyadenylation signal
  • bGH bovine growth hormone
  • a “host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector.
  • an exogenous polynucleotide such as a recombinant plasmid or vector.
  • genetically altered cells the term refers both to the originally altered cell and to the progeny thereof.
  • the prokaryotes contemplated in the present invention may include Avibacterium, Brucella, Escherichia coli, Haemophilus (e.g., Haemophilus suis ), Salmonella (e.g., Salmonella enteridis, Salmonella typhimurium, Salmonella infantis ), Lactococcus lactis, Lactobacillus, Shigella, Pasteurella , and Rimeirella.
  • Avibacterium Brucella
  • Escherichia coli e.g., Haemophilus suis
  • Salmonella e.g., Salmonella enteridis, Salmonella typhimurium, Salmonella infantis
  • Lactococcus lactis e.g., Lactococcus lactis, Lactobacillus, Shigella, Pasteurella , and Rimeirella.
  • a number of expression vectors may be selected.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as PBLUESCRIPT (Stratagene) and pET vectors (Novagen); piN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and the like; PGEX Vectors (Promega, Madison, Wis.).
  • the cell lines may be yeast (such as Saccharomyces cerevisiae, Pichia pastoris ), baculovirus cells, mammalian cells, plant cells.
  • the expression vectors of eukaryotic systems include, but are not limited to, pVR1020 or pVT1012 vectors (Vical Inc., San Diego, Calif.), PichiaPink Vector (Invitrogen, CA, USA), pFasBac TOPO vector (Invitrogen).
  • the subject matter disclosed herein is directed to a method of producing or making an antigenic polypeptide or antigen.
  • the method comprises the steps of: i) linking a polynucleotide encoding the antigen to a polynucleotide encoding an E2 protein; ii) expressing the fusion protein in a host; and iii) isolating the fusion protein from the host.
  • the antigen is linked to the N-terminus of E2.
  • the E2 protein is from Geobacillus stearothermophilus.
  • the subject matter disclosed herein is directed to a method of vaccinating an ovine, bovine, caprine, or swine comprising administering to the ovine, bovine, caprine, or swine an effective amount of a vaccine which may comprise an effective amount of a recombinant or fusion or chimeric FMDV antigen or protein and a pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle.
  • the method comprises a single administration of a vaccine composition formulated with an emulsion according to the invention.
  • the immunological or vaccine composition comprises FMDV antigens, including polypeptides and VLPs (virus-like particles), while an alternate embodiment provides for a vaccine comprising FMDV-E2 fusion or chimeric proteins. Electron microscopy indicates the FMDV-E2 fusion or chimeric proteins produce FMDV-E2 particles that resemble authentic FMDV virions or VLPs, and so immunological or vaccine compositions according to the instant invention encompass those comprising FMDV-E2 particles.
  • the subject matter disclosed herein is directed to a method of vaccinating an ovine, bovine, caprine, or swine comprising administering to the ovine, bovine, caprine, or swine an ovine, bovine, caprine, or swine FMDV antigen.
  • the subject matter disclosed herein is directed to a method of eliciting an immune response comprising administering to the ovine, bovine, caprine, or swine a vaccine comprising an ovine, bovine, caprine, or swine FMDV antigen.
  • the subject matter disclosed herein is directed to a method of protecting and/or preventing diseases in an ovine, bovine, caprine, or swine comprising administering to the ovine, bovine, caprine, or swine a vaccine comprising an ovine, bovine, caprine, or swine FMDV antigen.
  • the administering may be subcutaneously or intramuscularly.
  • the administering may be needle free (for example Pigjet or Bioject).
  • a prime-boost regimen can be employed, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen.
  • the immunological composition or vaccine used in primary administration is different in nature from those used as a booster.
  • This administration protocol is called “prime-boost”.
  • a prime-boost according to the present invention can include a recombinant viral vector which is used to express an FMDV coding sequence or fragments thereof encoding an antigenic polypeptide or fragment or variant thereof.
  • the viral vector can express an FMDV gene or fragment thereof that encodes an antigenic polypeptide.
  • Viral vector contemplated herein includes, but not limited to, poxvirus [e.g., vaccinia virus or attenuated vaccinia virus, avipox virus or attenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC, TROVAC; see e.g., U.S. Pat. No. 5,505,941, U.S. Pat. No.
  • poxvirus e.g., vaccinia virus or attenuated vaccinia virus, avipox virus or attenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC, TROVAC; see e.g., U.S. Pat. No. 5,505,941, U.S. Pat. No
  • avipox expression vector may be a canarypox vector, such as, ALVAC.
  • the avipox expression vector may be a fowlpox vector, such as, TROVAC.
  • the FMDV antigen of the invention to be expressed is inserted under the control of a specific poxvirus promoter, e.g., the entomopoxvirus Amsacta moorei 42K promoter (Barcena, et al.
  • the avipox expression vector may be a canarypox vector, such as, ALVAC.
  • the FMDV antigen, epitope or immunogen may be FMDV P1-3C.
  • the viral vector for expressing FMDV antigen may be a canarypox virus such as vCP2186, vCP2181, or vCP2176, or a fowlpox virus such as vFP2215 (see U.S. Pat. No. 7,527,960), or an adenovirus (US2016/0220659).
  • a prime-boost according to the present invention may include a recombinant FMDV antigen or protein produced in plants or algae (see US 2011/0236416), or insect cells (US2016/0220659).
  • a composition comprising the FMDV antigen of the invention is administered followed by the administration of vaccine or composition comprising a recombinant viral vector that contains and expresses the FMDV antigen in vivo, or an inactivated viral vaccine or composition comprising the FMDV antigen, or a DNA plasmid vaccine or composition that contains or expresses the FMDV antigen, or a recombinant FMDV antigen produced in plants, algae or insect cells.
  • a prime-boost protocol may comprise the administration of vaccine or composition comprising a recombinant viral vector that contains and expresses an FMDV antigen in vivo, or an inactivated viral vaccine or composition comprising an FMDV antigen, or a DNA plasmid vaccine or composition that contains or expresses an or FMDV antigen, or a recombinant FMDV antigen produced in plants, algae or insect cells, followed by the administration of a composition comprising the FMDV antigen of the invention.
  • both the primary and the secondary administrations may comprise the composition comprising the FMDV antigen of the invention
  • a prime-boost protocol comprises at least one prime-administration and at least one boost administration using at least one common polypeptide and/or variants or fragments thereof.
  • the vaccine used in prime-administration may be different in nature from those used as a later booster vaccine.
  • the prime-administration may comprise one or more administrations.
  • the boost administration may comprise one or more administrations.
  • compositions for target species that are mammals e.g., the dose volume of ovine, bovine, caprine or swine compositions, based on viral vectors, e.g., non-poxvirus-viral-vector-based compositions, is generally between about 0.1 to about 5.0 ml, between about 0.1 to about 3.0 ml, and between about 0.5 ml to about 2.5 ml.
  • the efficacy of the vaccines may be tested about 2 to 4 weeks after the last immunization by challenging animals, such as ovine, bovine, caprine or swine, with a virulent strain of FMDV, advantageously the FMDV 01 Manisa, 01 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran'96, A22 Iraq, SAT2 Saudi Arabia strains.
  • challenging animals such as ovine, bovine, caprine or swine
  • Still other strains may include FMDV strains A10-61, A5, A12, A24/Cruzeiro, C3/Indaial, O1, C1-Santa Pau, C1-05, A22/550/Azerbaijan/65, SAT1-SAT3, A, A/TNC/71/94, A/IND/2/68, A/IND/3/77, A/IND/5/68, A/IND/7/82, A/IND/16/82, A/IND/17/77, A/IND/17/82, A/IND/19/76, A/IND/20/82, A/IND/22/82, A/IND/25/81, A/IND/26/82, A/IND/54/79, A/IND/57/79, A/IND/73/79, A/IND/85/79, A/IND/86/79, A/APA/25/84, A/APN/41/84, A/APS/44/05, A/APS/50/05, A/APS/55/05, A/APS/66/05,
  • FMDV strains may be found on the European Bioinformatics Information (EMBL-EBI) web pages.
  • EBL-EBI European Bioinformatics Information
  • Both homologous and heterologous strains are used for challenge to test the efficacy of the vaccine.
  • the animal may be challenged intradermally, subcutaneously, spray, intra-nasally, intra-ocularly, intra-tracheally, and/or orally.
  • the prime-boost administrations may be advantageously carried out 1 to 6 weeks apart.
  • a semi-annual booster or an annual booster advantageously using the viral vector-based vaccine, is also envisaged.
  • compositions comprising the recombinant antigenic polypeptides of the invention used in the prime-boost protocols are contained in a pharmaceutically or veterinarily acceptable vehicle, diluent, adjuvant, or excipient.
  • the protocols of the invention protect the animal from ovine, bovine, caprine or swine FMDV and/or prevent disease progression in an infected animal.
  • the present invention contemplates at least one administration to an animal of an efficient amount of the therapeutic composition made according to the invention.
  • the animal may be male, female, pregnant female and newborn. This administration may be via various routes including, but not limited to, intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration.
  • the therapeutic composition according to the invention can also be administered by a needleless apparatus (as, for example with a Pigj et, Dermoj et, Biojector, Avij et (Merial, GA, USA), Vetj et or Vitaj et apparatus (Bioject, Oregon, USA)).
  • Another approach to administering plasmid compositions is to use electroporation (see, e.g. Tollefsen et al., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT Application No. WO99/01158).
  • the invention provides for the administration of a therapeutically effective amount of a formulation for the delivery and expression of an FMDV antigen or epitope in a target cell. Determination of the therapeutically effective amount is routine experimentation for one of ordinary skill in the art.
  • the formulation comprises an expression vector comprising a polynucleotide that expresses an FMDV antigen or epitope and a pharmaceutically or veterinarily acceptable carrier, vehicle, advjuant or excipient.
  • the pharmaceutically or veterinarily acceptable carrier, vehicle, adjuvant or excipient facilitates transfection or other means of transfer of polynucleotides to a host animal and/or improves preservation of the vector or protein in a host.
  • the subject matter disclosed herein provides a detection method for differentiation between infected and vaccinated animals (DIVA).
  • the use of the vaccine or composition of the present invention allows the detection of FMDV infection in an animal. It is disclosed herein that the use of the vaccine or composition of the present invention allows the detection of the infection in animals by differentiating between infected and vaccinated animals (DIVA).
  • DIVA infected and vaccinated animals
  • a method is disclosed herein for diagnosing the infection of FMDV in an animal using FMDV immunogenic detection method, such as, ELISA.
  • the subject matter disclosed herein is directed to a kit for performing a method of eliciting or inducing an immune response which may comprise any one of the recombinant FMDV immunological compositions or vaccines, or inactivated FMDV immunological compositions or vaccines, recombinant FMDV viral compositions or vaccines, and instructions for performing the method.
  • kits for performing a method of inducing an immunological or protective response against FMDV in an animal comprising a composition or vaccine comprising an FMDV antigen of the invention and a recombinant FMDV viral immunological composition or vaccine, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
  • kits for performing a method of inducing an immunological or protective response against FMDV in an animal comprising a composition or vaccine comprising an FMDV antigen of the invention and an inactivated FMDV immunological composition or vaccine, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
  • kits for prime-boost vaccination may comprise at least two vials: a first vial containing a vaccine or composition for the prime-vaccination according to the present invention, and a second vial containing a vaccine or composition for the boost-vaccination according to the present invention.
  • the kit may advantageously contain additional first or second vials for additional primo-vaccinations or additional boost-vaccinations.
  • a pharmaceutically or veterinarily acceptable carrier or vehicle or adjuvant or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer.
  • Other pharmaceutically or veterinarily acceptable carrier or vehicle or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone.
  • the pharmaceutically or veterinarily acceptable carrier or vehicle or adjuvant or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); the carrier, adjuvant, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.
  • the cationic lipids containing a quaternary ammonium salt which are advantageously but not exclusively suitable for plasmids, are advantageously those having the following formula:
  • R1 is a saturated or unsaturated straight-chain aliphatic radical having 12 to 18 carbon atoms
  • R2 is another aliphatic radical containing 2 or 3 carbon atoms
  • X is an amine or hydroxyl group, e.g. the DMRIE.
  • the cationic lipid can be associated with a neutral lipid, e.g. the DOPE.
  • DMRIE N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium; WO96/34109
  • DOPE dioleoyl-phosphatidyl-ethanol amine
  • the DMRIE:DOPE molar ratio is about 95:about 5 to about 5: about 95, or about 1:about 1, e.g., 1:1.
  • the DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be between about 50:about 1 and about 1:about 10, such as about 10:about 1 and about 1:about 5, and about 1:about 1 and about 1:about 2, e.g., 1:1 and 1:2.
  • pharmaceutically or veterinarily acceptable carrier, adjuvant, excipient, or vehicle may be a water-in-oil emulsion.
  • suitable water-in-oil emulsions include oil-based water-in-oil vaccinal emulsions which are stable and fluid at 4° C.
  • aqueous phase containing in total or in part a non-metabolizable oil (e.g., mineral oil such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil, or fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % of surfactants, preferably from 3 to 8 p/v %, the latter being in total or in part, or in a mixture either polyglycerol esters, said polyglycerol esters being preferably polyglycerol (poly)ricinoleates, or polyoxyethylene ricin oils or else hydrogenated polyoxyethylene ricin oils.
  • a non-metabolizable oil e.g., mineral oil such as paraffin oil
  • metabolizable oil e.g., vegetable oil, or fatty acid, polyol or alcohol esters
  • surfactants examples include ethoxylated sorbitan esters (e.g., polyoxyethylene (20) sorbitan monooleate (TWEEN 80®), available from AppliChem, Inc., Cheshire, Conn.) and sorbitan esters (e.g., sorbitan monooleate (SPAN 80®), available from Sigma Aldrich, St. Louis, Mo.).
  • sorbitan esters e.g., polyoxyethylene (20) sorbitan monooleate (TWEEN 80®), available from AppliChem, Inc., Cheshire, Conn.
  • sorbitan esters e.g., sorbitan monooleate (SPAN 80®), available from Sigma Aldrich, St. Louis, Mo.
  • the antigen-containing aqueous phase comprises a saline solution comprising one or more buffering agents.
  • the water-in-oil emulsion may be a water/oil/water (W/O/W) triple emulsion (U.S. Pat. No. 6,358,500). Examples of other suitable emulsions are described in U.S. Pat. No. 7,371,395.
  • the immunological compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants.
  • Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M.
  • cation lipids containing a quaternary ammonium salt e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.
  • a quaternary ammonium salt e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.
  • the oil in water emulsion (3) can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters.
  • light liquid paraffin oil European pharmacopoeia type
  • isoprenoid oil such as squalane, squalene
  • oil resulting from the oligomerization of alkenes e.g. isobutene or decene
  • the oil is used in combination with emulsifiers to form an emulsion.
  • the emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.
  • Some of the emulsions, such as TS6, TS7, TS8 and TS9 emulsions are described in U.S. Pat. No. 7,608,279 and U.S. Pat. No. 7,371,395.
  • the polymers of acrylic or methacrylic acid (1) are preferably crosslinked, in particular with polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996).
  • One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms.
  • the preferred radicals are those containing 2 to 4 carbon atoms, e.g.
  • the unsaturated radicals can also contain other substituents, such as methyl.
  • Products sold under the name Carbopol are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to Carbopol 974P, 934P and 971P.
  • the EMATM copolymers which are copolymers of maleic anhydride and of ethylene, which are linear or crosslinked, for example crosslinked with divinyl ether, are preferred.
  • the concentration of polymers of acrylic or methacrylic acid or of anhydride maleic and alkenyl copolymers in the final vaccine composition will be from 0.01% to 1.5% W/V, more particularly from 0.05 to 1% W/V, preferably from 0.1 to 0.4% W/V.
  • the adjuvant may include TS6 (U.S. Pat. No. 7,371,395), LR2, LR3 and LR4 (U.S. Pat. No. 7,691,368), TSAP (US20110129494), TRIGENTM (Newport Labs), synthetic dsRNAs (e.g. poly-IC, poly-ICLC [HILTONOL®]), and MONTANIDETM adjuvants (W/O, W/O/W, O/W, IMS and Gel; all produced by SEPPIC).
  • TS6 U.S. Pat. No. 7,371,395
  • LR2, LR3 and LR4 U.S. Pat. No. 7,691,368
  • TSAP US20110129494
  • TRIGENTM Newport Labs
  • synthetic dsRNAs e.g. poly-IC, poly-ICLC [HILTONOL®]
  • MONTANIDETM adjuvants W/O, W/O/W, O/W, IMS and
  • the cytokine or cytokines (5) can be in protein form in the immunological or vaccine composition, or can be co-expressed in the host with the immunogen or immunogens or epitope(s) thereof. Preference is given to the co-expression of the cytokine or cytokines, either by the same vector as that expressing the immunogen or immunogens or epitope(s) thereof, or by a separate vector thereof.
  • the invention comprehends preparing such combination compositions; for instance by admixing the active components, advantageously together and with an adjuvant, carrier, cytokine, and/or diluent.
  • Cytokines that may be used in the present invention include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon ⁇ (IFN ⁇ ), interferon ⁇ (IFN ⁇ ), interferon ⁇ , (IFN ⁇ ), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor ⁇ (TNF ⁇ ), tumor necrosis factor ⁇ (TNF ⁇ ), and transforming growth factor ⁇ (TGF ⁇ ).
  • G-CSF granulocyte colony
  • cytokines can be co-administered and/or sequentially administered with the immunological or vaccine composition of the present invention.
  • the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, a swine cytokine for preparations to be administered to swine).
  • the immunological composition and/or vaccine according to the invention comprise or consist essentially of or consist of an effective quantity to elicit a therapeutic response of one or more in vitro expressed polypeptides as discussed herein; and, an effective quantity can be determined from this disclosure, including the documents incorporated herein, and the knowledge in the art, without undue experimentation.
  • composition or vaccine may contain a dose from about 10 2 to about 10 20 , about 10 3 to about 10 18 , about 10 4 to about 10 16 , about 10 5 to about 10 12 VLPs (virus like particles) produced in vitro or in vivo from a viral vector, a plasmid, or baculovirus.
  • VLPs virus like particles
  • the viral vector may be titrated based on any virus titration methods including, but not limited to, FFA (Focus Forming Assay) or FFU (Focus Forming Unit), TCID 50 (50% Tissue Culture Infective Dose), PFU (Plaque Forming Units), and FAID 50 (50% Fluorescent Antibody Infectious Dose), and the VLPs produced in vitro (such as plasmid or baculovirus) can be titrated by hemagglutination assay, ELISA, and electron microscopy. Other methods may also be applicable depending on the type of VLP.
  • FFA Fluorescent Antibody Infectious Dose
  • the dose volumes can be between about 0.1 and about 10 ml, between about 0.2 and about 5 ml.
  • the FMDV VP1 gene from O1 Manisa serotype (SEQ ID NO:1 encoding SEQ ID NO:2) was amplified by PCR and cloned into pET30b vector (Novagen).
  • the resulting plasmid pPX216 contains the FMDV-E2 fusion which has the FMDV VP1 gene at the 5′end and the core domain of E2 gene (SEQ ID NO:3 encoding SEQ ID NO:4) at the 3′ end.
  • the FMDV VP1 gene from A24 serotype (SEQ ID NO:7 encoding SEQ ID NO:8) was amplified by PCR and cloned into pET30b vector (Novagen).
  • the resulting plasmid pPX236 contains the FMDV-E2 fusion which has the FMDV VP1 gene at the 5′end and the core domain of E2 gene (SEQ ID NO:3 encoding SEQ ID NO:4) at the 3′ end.
  • the FMDV VP1 gene from Asia Shamir serotype (SEQ ID NO:13 encoding SEQ ID NO:14) was amplified by PCR and cloned into pET30b vector (Novagen).
  • the resulting plasmid pPX237 contains the FMDV-E2 fusion which has the FMDV VP1 gene at the 5′end and the core domain of E2 gene (SEQ ID NO:3 encoding SEQ ID NO:4) at the 3′ end.
  • lysates were incubated 15 minutes at 4° C. with 1 ⁇ L of Benzonase Nuclease (Novagen)/per liter of culture. 10 ⁇ L of proteins from lysates were analyzed to determine the expression level by SDS-PAGE 4-12% Bistris electrophoresis followed by Coomassie blue staining and western blot analysis. For each condition, 2 clones were analyzed.
  • the FMDV-E2 fursion protein was produced as insoluble fraction without His tag and further purified by IEX and SEC, prior to being refolded.
  • the Western blot and dot blot have shown that the FMDV-E2 fusion protein produced the correct size and the expressed FMD-E2 reacted with swine serum vaccinated with FMDV vaccines.
  • FIGS. 4 and 5 The physical and biochemical characterization of the FMDV-E2 nanoparticles was carried out as shown in FIGS. 4 and 5 .
  • the expressed antigens were checked by electronic microscopy for the presence of nanoparticles structures.
  • FIG. 4A shows the particles of E2 alone protein.
  • FIG. 4B shows the particles of E2-FMDV-E2 fusion protein.
  • FIG. 4C shows the particles of cdE2-FMDV protein which contains the mixture of stand-alone E2 protein and FMDV-E2 fusion protein in the 2:1 ratio.
  • the objective of the study was to assess the immunogenicity in conventional pigs, of FMD 01 Manisa vaccine.
  • the vaccine was tested in TS6 and polyacrylic acid polymer formulations. Each formulation was administered in two injections three weeks apart (D0-D21) to pigs (see Table 2). Pigs were 8-9 weeks of age on D0. Immunogenicity was assessed through serology study and cell mediated immunity (CMI) assays (D27, D42) as outlined in Table 3.
  • CMI cell mediated immunity
  • the parameters monitored include: bodyweight on D1 for randomization and D42 before euthanasia, serology on D1, D20 and D42, CMI assay on D27 and D42, clinical signs (general reactions from D1 to D21 and D21 to D23), and local reactions at injection sites (D0 to D2 and D21 to D23).
  • FIGS. 6 and 7 have shown that specific plasma cells were detected in all FMDV-E2 groups with very weak response for G1 (FMDV alone). Better responses were obtained with G2 compared to G3 and G4. G2 group showed prominent responses using the FMDV-E2 coating.
  • FIGS. 8 and 9 have shown that specific IFN ⁇ responses were detected for all vaccinated groups using any re-stimulation with highest IFN ⁇ response detected using the FMDV-E2 re-stimulation for all groups.
  • FIG. 10 shows the results after 7 days re-stimulation with protein FMDV-E2 (number of antibody secreting memory B cells). The results have shown that specific B cells were detected for all FMDV-E2 groups and weak response for G1 (FMDV alone). Better responses were obtained with G2 compared to G3 and G4. Lower or no responses were obtained using coating FMDV peptide and AI O1M. Best results were obtained in groups G2 and G3 using FMDV-E2 coating.
  • the O1M vaccines induced humoral and cellular responses with strong, serotype-specific, neutralizing antibody responses, consistent proportion of specific IFN ⁇ responses, presence of memory B cells, indicating good levels of protection against homologous and heterologous FMDV infections.
  • the efficacy of the products under test was assessed serologically by FMDV seroneutralisation test and immunologically through cellular immune response assays. The best results were obtained with the FMDV-E2 particles adjuvanted with TS6.
  • the objective of the study was to assess the immunogenicity in conventional pigs, of A24 Cruzeiro and Asia1 Shamir vaccines.
  • the protective efficacy induced by the composition or vaccine is evaluated against the FMDV pathogen by vaccination challenge in animals.
  • the protective effect is evaluated by clinical observations and/or viral load of the specific pathogen in tissues and blood.
  • the blood samples from the vaccinated animals are taken at various stages and tested for serology and CMI assays. The results show that the composition or vaccine of the present invention is immunogenic and provides protection in animals.

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